1. Field of the Invention
The present invention relates to a flow cytometry apparatus, and more particularly, concerns a flow cytometry apparatus for determining one or more characteristics of cells or the like which includes improved optics for wide angle light scatter and fluorescence detection.
2. Description of the Prior Art
Flow cytometry apparatuses rely upon the flow of cells or other particles in a liquid flow stream in order to determine one or more characteristics of the cells under investigation. For example, a liquid sample containing cells is directed through the flow cytometry apparatus in a rapidly moving liquid stream so that each cell passes serially, and substantially one at a time, through a sensing region. Cell volume may be determined by changes in electrical impedance as each cell passes through the sensing region. Similarly, if an incident beam of light is directed at the sensing region, the passing cells scatter such light as they pass therethrough. This scattered light has served as a function of cell shape, index of refraction, opacity, roughness and the like. Further, fluorescence emitted by labeled cells which have been excited as a result of passing through the excitation energy of the incident light beam is detectable for identification of specifically labeled cells. Not only is cell analysis performed on the flow cytometry apparatuses, but sorting of cells may also be achieved. Lasers have been used as the source of the incident beam of illumination in flow cytometry apparatuses, as well as sources of incoherent and non-collimated light, such as mercury or xenon arc lamps.
In most of the presently known and available flow cytometry apparatuses, fluorescence emitted by the cells is typically collected at an angle whose viewing axis is 90.degree. relative to the axis of light excitation. In addition, wide angle light scatter, typically at 90.degree., has been found to obtain information about cells relating to shape and internal morphology. Inasmuch as both light scatter and fluorescence at 90.degree. may be collected with the same optical collection system, filters or light beam separators have been utilized to split the 90.degree. fluorescence and light scatter so that each may be detected separately. Furthermore, for most efficient collection, and if the incident light beam is provided by a laser, the light beam is typically polarized with the electric vector oriented normal to the plane containing the 90.degree. angle defined by the excitation and collection axes.
It is a property of scattering, that light scattered at right angles from the incident beam retains its polarization. On the other hand, the polarization of fluorescence depends upon the molecules themselves, and not upon the incident light beam. In practice, however, fluorescence from most cells is usually modestly unpolarized.
At present, dichroic filters are employed in separating a light beam having both fluorescence and scatter components. Such filters frequently do not transmit fluorescence as efficiently as would be desirable. Additionally, dichroic filters are typically wavelength dependent. For instance, changing the excitation wavelength from one frequency to another would require a different dichroic filter. It is also expensive to fabricate these types of dichroic filters.
In some instances, dielectric broadband filters have been employed to separate the light scatter and fluorescence components of a light beam. Such broadband filters normally do not have polarization benefits. In other words, and for example, reflecting 25% of the scatter would imply transmitting only 75% of the fluorescence.
It has also been known to employ uncoated glass or quartz as a beam splitter plate in a flow cytometry apparatus. Such a glass plate has typically been positioned at a 45.degree. angle relative to the incident light beam comprising both light scatter and fluorescence components. At the 45.degree. angle, fluorescence is transmitted efficiently, but light scatter is reflected rather inefficiently.
Many different flow cytometry apparatuses and techniques have been described in a review article by John A. Steinkamp, entitled "Flow Cytometry," Review of Scientific Instruments 55 (9), September 1984. While there are a variety of different approaches for optically sensing the characteristics of cells, there is still a need for further improvements in such optics so that cellular information may be obtained more efficiently and reliably.